Diagnosis of complex diseases and response to treatments are often associated with multiple biomolecules rather than a single identifiable biomarker. In contrast to conventional techniques which measure one analyte at a time, multiplexing technologies may measure, within a single assay, tens to thousands of different biomolecules, e.g., proteins or nucleic acids, from a single biological sample using identical conditions. Basically, capture molecules of different types, e.g. antibodies, target proteins, peptides or nucleic acids, are provided in an assay apparatus filled with the sample, each type of capture molecule being designed to form with the biomarker to be searched in the sample a particular fluorescently labeled complex (usually referred as “the targeted fluorescent labeled biomarker”). One main issue of multiplexing techniques is to determine during the single assay the fluorescence originating from a single type of fluorescently labeled biomarkers bound to their complementary capture molecules (hereafter “individual fluorescence”).
Various multiplexing technologies are available, each being usually classified according to its specific encoding strategy addressing this issue. The most popular commercially available multiplexing technologies are array-based and bead-based. In array-based technologies, capture molecules are bound on a panel in a known arrangement to form a 2D array of “capture spots”, each dedicated to the capture of a particular biomarker. Such planar arrays thus rely on x-y-coordinates of the capture spots to determine the fluorescence of each biomarker. Bead-based technologies are based on spectral encoding in which the color and intensity allows discriminating each bead population. They have proven to be highly flexible and scalable. However, the currently available bead-based systems are designed to run cost-effectively in batches and the need for waiting on sufficient samples to fill the plate can slow down the turnaround time in routine clinical testing. Furthermore, both technologies suffer from slow binding kinetics, as they are mainly driven by diffusion. This usually imposes long sample incubation times even when agitation is used to speed up the process. In addition, diffusion limited binding regime can also contribute to reported high intra- and interassay variations.
To address the above limitations, a multiplexing technology based on encoded microparticles and microfluidic channels has been designed. This technology is for example described in “Rapid, Sensitive and Real-Time Multiplexing Platform for the Analysis of Protein and Nucleic-Acid Biomarkers”, Didier Falconnet and al., Anal. Chem. 2015, 87, 1582-1589 and its supporting information downloadable from the website http://pubs.acs.org. One apparatus embodying this technology is sold under the reference named “Evaluation™” by MyCartis, Ghent, BE. The main components of this technology (encoded microparticles, microfluidic channel cartridge and instrumentation) are now briefly described in relation with FIG. 1.
Referring to FIG. 1A, the encoded microparticles 10, or “carriers”, are disk-shaped, 40 μm in diameter and 10 μm in height, and are produced from silicon wafers, e.g. using MEMS manufacturing technology. The periphery of each microparticle 10 is unambiguously encoded using a 10-digit binary code Idm 12, or “identifier”, formed by the presence or absence of holes 14. Capture molecules are bound to both faces of the microparticles 10, the latter thus acting as a solid support for a multitude of possible capture molecules, including antibodies, target proteins, peptides, nucleic acids or other biomolecules. More particularly, a single type of capture molecules (i.e. capture molecules dedicated to the capture of a particular biomarker) is tethered onto microparticles sharing the same code Idm.
Referring to FIG. 1B, the cartridge 20, or “assay plate”, features a plurality of microscale channels 22 able to host encoded microparticle mixes and thus enabling running multiple samples simultaneously or sequentially (i.e. at different dates). Each channel 22 is made of transparent walls and connects an inlet well 24 and an outlet well 26 which enable the pressurization of the channel 22 above the atmospheric pressure, enabling the microfluidic operation of the channel. The channels 22 are at least 5 times, e.g. 10 times, wider than the microparticles' diameter, and include each a filter structure to restrain the microparticles in a detection zone of the channel. The height of the channels is optimized for efficient microparticle loading and tiling. The shallow channel height prevents microparticles from overlapping each other so that microparticles are arranged in the channel in a monolayer arrangement with one of their faces fully acquirable for imaging purpose. This monolayer arrangement of microparticles in the channel thus enables the use of high resolution imaging for both decoding and fluorescence quantification. Each channel can be loaded with up to thousands of microparticles, a fully loaded channel enabling a multiplexing of more than one hundred different biomarkers with tens of microparticles for each biomarker to be searched for. For multiplexing analysis purpose, a microparticle mix loaded in a channel includes multiple microparticles sharing an identical code, thereby forming a population which provides measurement redundancy for statistical confidence.
Instrumentation aims at acquiring images of the channel, at controlling the fluid actuation and the temperature in the channels and at analyzing acquired images. More particularly, instrumentation comprises:
an optical system 28, with for example a long-working distance objective and a high-sensitivity CMOS camera, to acquire bright field and epi-fluorescence images (e.g. excitation at 640 nm, e.g. using a laser) of the detection zone of the cartridge. The objective is mounted on an automated x-y-z stage for scanning each channel either during the assay for real-time readout or in end point;
lighting system (not illustrated) for uniformly illuminate the detection zone in order to get a high contrast image of the microparticles in bright background for decoding purpose;
a controlling unit (not illustrated) for controlling the microfluidic operation of the cartridge (inlet/outlet wells, pressurization, temperature . . . ), as well as the operation of the optical system;
a computing unit 30 (e.g. personal computer, a server, or more generally any computing hardware configured to receive data from the camera and process the data according to instructions stored on a memory . . . ), possibly part of the controlling unit or independent therefrom, coupled to the camera for receiving images thereof and running an multiplexing analysis computing program to automatically quantify the biomarkers in the tested sample based on the fluorescent and bright-light images acquired by the optical system.
Referring to FIGS. 2A-2E, a multiplex analysis embodied by the aforementioned multiplexing technology thus consists in:
producing a suspension mix of microparticles 32, the microparticles 10 being chosen based on the biomarkers to be searched for in a tested sample and loading the liquid mix of microparticles 32 in the cartridge 20 to fill one or more channels 22 so that a planar arrangement of the microparticle is provided for optimal readout purpose (FIG. 2A);
loading the tested liquid sample, along with, if required, reagents (e.g. for sandwich reactions), in the channels 22, thereby initiating incubation and/or binding reaction in microfluidic environment between the biomarkers 34 in the tested sample and the capture molecules 36 bound to the microparticles 10 (FIG. 2B);
acquiring images of the detection zone of the channels 22 (e.g. in real time or once at the end of the process). While the microparticles 10 are immobilized in the channels 22 thanks to pressurization and filter elements, a cycle of image acquisition preferably consists in acquiring consecutively a bright field image 38 and a fluorescence image 40 or vice versa, so that the position of each microparticles is the same in both images (FIG. 2C);
for each channel 22 and for each pair of bright field and a fluorescence images 38, 40 of the channel:
analyzing the bright field image 38 to identify location Xi of each microparticle 10 in the channels 22 and to read the code Idm 12 of each microparticle 10;
analyzing the fluorescence image 40 to determine the fluorescence φmeas of each microparticle 10 in the fluorescent image (e.g. corresponding to the maximum of a kernel density fitted on the pixels of the portion of the image corresponding to the central portion of the microparticle);
for each channel 22 and each population of microparticles 10 sharing the same code Idm in the channel:
computing an aggregate value of fluorescence φpop for the population, e.g.: applying a Tukey's boxplot filter on fluorescences φmeas to filter out abnormal fluorescence values φmeas and thereafter computing the aggregate value as the arithmetic mean value of the remaining fluorescences φmeas (FIG. 2D);
determining the biomarker concentration [b] in the tested sample (or “titration”) based on the aggregate value φpop using a stored relationship fluorescence versus concentration (e.g. table, analytic mathematical model, . . . ) determined beforehand for the biomarker and stored in a digital memory of the computing unit 30 (FIG. 2E).
The computed concentrations are then displayed to the user and/or stored on a digital memory (e.g.: the one of the computing unit).
This multiplexing technology allows (i) short assay times and high reproducibility thanks to reaction-limited binding regime, (ii) dynamic control of assay conditions and real-time binding monitoring allowing optimization of multiple parameters within a single assay run, (iii) compatibility with various immunoassay formats such as co-flowing the samples and detection antibodies simultaneously and hence simplifying workflows, (iv) analyte quantification based on initial binding rates leading to increased system dynamic range and (v) high sensitivity via enhanced fluorescence collection, (vi) opportunity to run monoplex (i.e. providing only one type of capture molecules for the quantitative measure of a particular biomarker) assay if desired.
However, in some instances, one observes divergence between a biomarker concentration [b] which is computed from multiplex assay data and the biomarker concentration [b] computed from monoplex assay data.